Matrix‐assisted laser desorption ionization for rapid determination of the sequences of biologically active peptides isolated from support‐bound combinatorial peptide libraries

Youngquist, R.S.; Fuentes, Gary R.; Lacey, Martin P.; Keough, T.; Baillie, Thomas A.

doi:10.1002/rcm.1290080115

Cited by 61 publications

(37 citation statements)

References 15 publications

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“…Index libraries using electrophoretic tags have been successfully employed and peptides as well as non-peptidic structures have been synthesized by this method [10,13,18]. Alternative structure determination can also be accomplished using MS-MALDI TOF, where small amounts of deletion peptides are generated during synthesis by simple capping procedure and subsequently analysed after release from beads [43]. This technique is, however, not suitable for the analysis of motiflibraries since these libraries contain multiple structures on each bead and consequently determination of the mass difference between deletion peptides is unfeasible.…”

Section: Structure Determinationmentioning

confidence: 99%

Synthesis and Screening of an Indexed Motif-Library Containing Non-proteinogenic Amino Acids

Østergaard¹,

Holm²

1997

J. Peptide Sci.

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In an effort to increase the probability of finding novel peptides in resin-bound combinatorial libraries displaying affinity to various macromolecular targets, we increased the diversity of a solid-phase library considerably by synthesizing multiple structures on each bead - a motif-library - including 45 building blocks. The building blocks consist of L-aa, D-aa and eight hydrophobic non-proteinogenic alpha-amino acids. A library with the format O-Z0-1-O-Z0-1-O-XX-resin was synthesized giving the four motifs OOOXX, OZOOXX, OOZOXX, OZOZOXX corresponding to 364.500 different motifs (45(3) x 4 theoretical combinations). The positions O are defined amino acids while Z represents three mixtures pi, omega, phi, where pi is a mixture of polar and charged residues, omega is a mixture of aliphatic residues and phi is a mixture of aromatic residues. X represents a mixture of all 45 residues. The library was screened with the macromolecular target streptavidin which served as a model receptor. Binding peptides were sequenced by microsequencing. We included small amounts of norvaline and norleucine in the library, which served as index residues to be able to distinguish between LD-amino acids and other residues with the same retention time in the HPLC system. Beads that interact with the receptor were found, and the binding motifs that appeared had no homology to known binding motifs found in either L-aa or D-aa libraries, instead motifs with the non-proteinogenic residues L-phenylglycine, O-benzyl-L-hydroxyproline and O-benzyl-L-tyrosine dominated. The novel peptides inhibit binding of biotin to streptavidin but do not bind to avidin, and the affinity is higher than the peptides found in linear all L-aa peptide libraries.

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Section: Structure Determinationmentioning

confidence: 99%

Synthesis and Screening of an Indexed Motif-Library Containing Non-proteinogenic Amino Acids

Østergaard¹,

Holm²

1997

J. Peptide Sci.

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“…Both the synthetic and random peptides are assayed for their ability to bind to the antibody, allowing identification of the epitope. Recently, matrix-assisted laser desorption mass spectrometry has been incorporated into this approach to allow rapid sequence determination of the synthetic peptides that bind the antibody (Youngquist et al, 1994). An unfortunate drawback to these rapid techniques is that only continuous epitopes are typically identified.…”

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confidence: 99%

Epitope mapping of the gastrin‐releasing peptide/anti‐bombesin monoclonal antibody complex by proteolysis followed by matrix‐assisted laser desorption ionization mass spectrometry

1994

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We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDIITOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDIITOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDUTOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDIITOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

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“…An extension that allows wider chemical diversity of the libraries is to "encode" the library in some way. Brenner and Lerner (6) proposed encoding each molecule of the library with an oligodeoxynucleotide, which could be used both for identification and for the enrichment of active members, and the chemistry for this has been implemented by Janda and coworkers (7).Others have encoded (8)(9)(10)(11) …”

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confidence: 99%

“…Others have encoded (8)(9)(10)(11) beads, each carrying a single component of the chemical library, with peptides, oligonucleotides, and organohalide tags. The bead with the active component must be isolated and then the tag must be analyzed by mass spectrometry (8) or Edman degradation (9) for peptide analysis, by electron-capture gas chromatography for organohalide tags (10), or by PCR for deoxynucleotide tags (11).…”

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Recursive deconvolution of combinatorial chemical libraries.

Erb

Janda

Brenner

1994

Proc. Natl. Acad. Sci. U.S.A.

103

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A recursive strategy that solves for the active members of a chemical library is presented. A pentapeptide library with an alphabet of Gly, Leu, Phe, and Tyr (1024 members) was constructed on a solid support by the method of split synthesis. One member of this library (NH2-Tyr-Gly-GlyPhe-Leu) is a native binder to a -endorphin antibody. A variation of the split synthesis approach is used to build the combinatorial library. In four vials, a member of the library's alphabet is coupled to a solid support. After each coupling, a portion of the resin from each of the four reaction vials was set aside and catalogued. The solid support from each vial is then combined, mixed, and redivided. The steps of (i) coupling, (i) saving and cataloging, and (iii) randomizing were repeated until a pentapeptide library was obtained. There is increasing interest in synthesizing large numbers of molecules in parallel and in analyzing these pools for members with biological activity. So called "irrational" drug design, involving selection from combinatorial libraries, is becoming accepted as a useful method of finding pharmacologically active compounds.The main difficulty with this approach is a way of finding the compounds with defined activity, especially when the libraries used are large. Peptides and oligonucleotides bound to an immobilized receptor can be eluted and directly sequenced (1-3). Although oligonucleotides can be amplified by PCR, peptides may require several runs to obtain sufficient material for analysis. Alternatively, peptides, synthesized on beads, have been identified by isolating beads that have bound a receptor and then sequencing the released peptide. Peptides have also been identified by synthesizing them in arrays or on small surfaces (4, 5).These methods are restricted by the chemistries involved in the synthesis or the analysis. An extension that allows wider chemical diversity of the libraries is to "encode" the library in some way. Brenner and Lerner (6) proposed encoding each molecule of the library with an oligodeoxynucleotide, which could be used both for identification and for the enrichment of active members, and the chemistry for this has been implemented by Janda and coworkers (7).Others have encoded (8)(9)(10)(11)

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Matrix‐assisted laser desorption ionization for rapid determination of the sequences of biologically active peptides isolated from support‐bound combinatorial peptide libraries

Cited by 61 publications

References 15 publications

Synthesis and Screening of an Indexed Motif-Library Containing Non-proteinogenic Amino Acids

Synthesis and Screening of an Indexed Motif-Library Containing Non-proteinogenic Amino Acids

Epitope mapping of the gastrin‐releasing peptide/anti‐bombesin monoclonal antibody complex by proteolysis followed by matrix‐assisted laser desorption ionization mass spectrometry

Recursive deconvolution of combinatorial chemical libraries.

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